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Fig. 1 | BMC Molecular Biology

Fig. 1

From: Signal transducer and activator of transcription STAT5 is recruited to c-Myc super-enhancer

Fig. 1

c-Myc gene expression in Ba/F3-derived cell lines. a STAT5 protein and phosphorylation levels in the parental Ba/F3 cells and in Ba/F3 cells stably expressing STAT5A-WT (Ba/F3-WT) and STAT5A-1*6 (Ba/F3-1*6). Ba/F3 and Ba/F3-WT cells, which grow in IL-3-containing medium, were withdrawn from IL-3 for 11 h and stimulated with IL-3 for 15 min. IL-3-independent Ba/F3-1*6 cells were stimulated with IL-3 in parallel. Brij whole-cell protein lysates (20 μg) were analysed by Western blot using antibodies specific for phosphorylated STAT5 (pSTAT5), total STAT5A and STAT5B (STAT5A/B), transgenic STAT5A-WT and STAT5A-1*6 (FLAG), and α-tubulin as a loading control. STAT5A/B signal in parental Ba/F3 cells corresponds to endogenous STAT5 protein levels, while the signals detected in the stable cell lines Ba/F3-WT and Ba/F3-1*6 represent both endogenous (STAT5A and STAT5B) and transgenic (STAT5A-WT or -1*6) proteins. b STAT5-mediated expression of c-Myc in Ba/F3-WT and -1*6 cells. Rested Ba/F3-WT cells stably expressing wild-type STAT5A were pre-treated 30 min with 200 nM TSA and further stimulated with IL-3 for 30 min. Ba/F3-1*6 cells expressing constitutively active STAT5A-1*6 were treated in parallel with 200 nM TSA for 60 min. Expression of STAT5 target genes (Cis, Osm, c-Myc) and of the housekeeping gene 36b4 was analysed by RT-qPCR. Expression of c-Myc, like that of Cis and Osm, was induced by STAT5A-WT and by constitutively active STAT5A-1*6, in an IL-3-dependent and -independent manner respectively. STAT5A-WT- and STAT5A-1*6-mediated expression of c-Myc, Cis and Osm was inhibited by the deacetylase inhibitor trichostatin A (TSA). Student’s t tests were employed to compare on the one hand IL-3-induced (WT) or 1*6-induced gene expression to the unstimulated WT control, and on the other hand TSA-treated to the vehicle control in each condition; **P < 0.01, ***P < 0.001, ****P < 0.0001; a P value <0.05 was considered statistically significant. c Expression of STAT5 target genes is inhibited by both deacetylase (TSA) and BET (JQ1) inhibitors. Ba/F3 cells were pre-treated with 20 nM TSA or 500 nM JQ1 for 30 min and stimulated with IL-3 for 60 min. Expression of STAT5 target genes (Cis, c-Myc) and of control genes (IL-3-dependent MAPK target gene JunB and housekeeping gene 36b4) was analysed by RT-qPCR, as above. IL-3-induced expression of Cis and c-Myc, but not that of JunB, was inhibited by TSA and JQ1. One-way ANOVA with Dunnett’s multiple comparison test was used to assess differences between TSA- or JQ1-treated conditions and the vehicle-treated IL-3-stimulated control; ***P < 0.001; a P < 0.05 was considered statistically significant

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